Therefore, experiments should be set up to test a number of different conditions simultaneously. Or purifying a MAb using the ÄKTA avant 25 system controlled by UNICORN 6 software.ĭoE for Screening Loading Conditions on Capto adhere: Conductivity, pH, and sample load are important parameters for sample loading on Capto adhere ( 1). We conducted the seven experiments summarized in Table 1 to develop the two-step method f An XK 50/20 column was packed with MabSelect SuRe for an optimized polishing step (step 4, Table 1). We selected prepacked MabSelect SuRe and Capto adhere columns in HiTrap (1 mL) and HiScreen (4.7 mL) formats for various experiment steps (Table 1). Capto adhere was used for all ion-exchange runs. We followed the same method for all affinity chromatography runs and used MabSelect SuRe. Runs made use of the system’s pressure–flow regulation feature, which prevents excessive pressure from building up during sample loading. Table 2: Design layout of the DoE for screening of loading conditions and results from the screening ()Ĭolumns, Equipment, and MAb Purification: We used the ÄKTA avant 25 system with UNICORN 6 software and two sample inlet valves for automated sample loading and wash. In DoE, multiple factors are varied simultaneously, and a statistical model is generated for process validation and to support decision making (Table 2). We performed screening and robustness studies using a design of experiments (DoE) application included in UNICORN 6 control software. Method development included determining elution pH (Figure 3) and dynamic binding capacity on MabSelect SuRe to address step 1 (Table 1) and screening of loading conditions on Capto adhere (Figure 5) followed by a robustness study for step 2 (Table 1). Feed was based on a clarified Chinese hamster ovary (CHO) cell supernatant, which was challenging because of the MAb’s low solubility. Using the newly developed ÄKTA avant 25 chromatography system, we demonstrated rapid method development for a two-step MAb purification process. Loading conditions therefore need to be optimized, with a trade-off made between yield and purity. Loading conditions are always a compromise between those favoring yield and those favoring contaminant clearance, especially for cases in which several contaminant species need to be removed. For example, conduct polishing using Capto adhere medium in flow-through mode under conditions that allow monomeric antibodies to pass directly through a column while contaminants are adsorbed. To take full advantage of the benefits of multimodal chromatography medium, you should thoroughly optimize your purification process. The high purity obtained after initial capture on a protein-A–based medium (e.g., MabSelect SuRe, typically >99%) combined with a multimodal ion-exchange medium (e.g., Capto adhere) for the polishing step can reduce a traditional three-step process to a highly productive two-step process. You can use various chromatography techniques and combinations of techniques (especially ion-exchange and hydrophobic interaction chromatography) to perform subsequent downstream processing.Ī multimodal chromatography medium - capable of anion exchange as well as other types of interactions - enables selective removal of antibody dimers and aggregates (D–A), host-cell proteins (HCPs), DNA, and viruses at the same time ( 1, 2). Protein A–based chromatography media are therefore the basis for a platform approach to MAb purification. Protein A is the ligand of choice for capture when purifying MAbs because of its high selectivity that provides excellent purity and its ease of use at large and small scale. KEY WORDS: PROCESS DEVELOPMENT, MULTIMODAL CHROMATOGRAPHY, DESIGN OF EXPERIMENTS (DoE), PLATFORM TECHNOLOGY WHO SHOULD READ: PLANT MANAGERS, PROCESS ENGINEERS, ANALYSTS With design of experiments (DoE) software incorporated, the ÄKTA avant 25 system can easily and quickly establish optimal chromatography conditions, further reducing process development time (Figure 2). However, we propose a more time-efficient two-step approach (Figure 1) using the ÄKTA avant 25 system for fully automated process development. Opportunities to eliminate manually intensive steps all support an enhanced development process.Ī typical monoclonal antibody (MAb) purification process includes three chromatographic purification steps. An ability to scientifically design product and process characteristics that meet specific objectives is crucial. Easy translation of experimental ideas into process steps and insight into the effects of changes in chromatography parameters both help speed development and contribute toward achieving quality by design (QbD) objectives. ![]() Time and flexibility are essential in purification process development for biopharmaceuticals.
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